The present invention relates to the regulation of gene expression in eukaryotic cells. More specifically, the present invention relates to a system for controlled transgene transcription using both chimeric activator and repressor proteins.
Expression of target genes with homo- and heterologous eukaryotic systems is widely used in biological and medical research, as well as biotechnology and somatic gene therapy. Recently, considerable progress has been made in the control of expression of target genes. Regulated gene expression has been achieved by the use of heterologous or artificial (chimerical) transcription factors responding to an exogenously added inducer drug which acts as a bona fide ligand. Typically, these transcription factors recognize cognate regulatory elements in the promoter of the target gene and the ligand regulates the interaction of the factor with the DNA or the interaction of the DNA-bound factor with a transcriptional activation domain.
Ideally, the administration or removal of the ligand results in a switch between the on or off states of activity of the target gene. Several small molecule ligands have been shown to mediate regulated gene expressions, either in tissue culture cells and/or in transgenic animal models. These include the FK1012 and rapamycin immunosupressive drugs [Spencer et al., Science 262: 1019-1024 (1993); Magari et al., J. Clin. Invest. 100: 2865-2872 (1997)], the progesterone antagonist mifepristone (RU486) [Wang, Proc. Natl. Acad. Sci. USA 93: 8180-8184 (1994); Wang et al., Nature Biotech 15: 239-243 (1997)], the tetracycline antibiotic derivatives [Gossen and Bujard, Proc. Natl. Acad. Sci USA 89: 5547-5551 (1992); Gossen et al., Science 268: 1766-1769 (1995); Kistner et al., Proc. Natl. Acad. Sci. 93: 10933-10938 (1996)], and the insect steroid hormone ecdysone [No et al., Proc. Natl. Acad. Sci USA 93: 3346-3351 (1996)].
One of the major drawbacks with most existing expression systems, however, is the lack of control over the expression level of these target genes which leads to a constitutive production of the encoded recombinant proteins. In most applications, it is desirable to limit the production of such proteins to a defined time window, and to a precisely controlled expression level. This is especially true for the wealth of somatic gene therapy projects including the generation of novel viral and non-viral vectors, or the biotechnological production of recombinant proteins which interfere with cell growth during the exponential phase of cell cultivation in bioreactors. Other examples are found in the analysis of finction and role of specific genes in development and homeostasis of an organism. Therefore, it is imperative to design novel expression systems, the activity of which is under tight genetic control. Such systems are required to exhibit a low (or no) level of transgene expression in the xe2x80x9coff-statexe2x80x9d and high levels of transgene expression in the xe2x80x9con-state.xe2x80x9d
Thus, a need remains in the art for a versatile, specific, and stringent genetic switch mechanisms to control target gene expression for the expression of cell-toxic proteins in vitro and for tight transgene regulation in transgenic animals and in gene therapy.
The present invention relates to a new transgene regulatory system that offers a low background activity when the gene is turned off, a wider window of regulation, and a fast response kinetic after addition of the inducer ligand. The genetic switch system relies on a regulatory network of at least one activator and at least one repressor of transcription, both acting to allow for a high induction window of target gene expression in response to the tetracycline, or a derivative thereof such as doxycycline (dox). Active repression of the transgene in the absence of the inducer drug results in an extremely low background expression level, while autoregulatory positive feedback loops ensure high levels of activated gene expression upon ligand administration. The tight regulation mediated by this regulatory system should prove useful for a variety of applications in biotechnology and medicine.